Survival of the bacterium depends on the health of its cell wall. Because of the indispensable nature of the cell wall, the biosynthetic enzymes of its assembly and the cell wall itself are targets of several of the existing antibiotics. The final steps of cell wall assembly and maturation are performed by a group of enzymes referred to as penicillin- binding proteins (PBPs) on the surface of the cytoplasmic membrane. As the name suggests, these enzymes are inhibited by beta-lactam antibiotics (e.g., penicillins), and they serve as validated targets for the bactericidal activity by these antibiotics. Despite their importance, many aspects of biochemistry of PBPs have not been investigated and there are currently no other known effective inhibitors for these enzymes. This project addresses this paucity of information within the scope of three Specific Aims. Specific Aim 1 deals with PBPs with the DD-transpeptidase activity. These enzymes perform the important cross-linking reaction of the cell wall. The studies build on earlier work from the Mobashery lab in elucidation of the microscopic steps in the enzymic reaction, both by experiments and by computational analyses. The knowledge is applied to a strategy for mechanism-based inhibition of these enzymes. Specific Aim 2 will address the mechanistic details of PBPs with the DD-carboxypeptidase activity by both experiments and computation. There enzymes process the cell wall hydrolytically, whereby they moderate the degree of cross-linking that is performed by DD-transpeptidases. Specific Aim 3 addresses the catalytic mechanisms of bifunctional PBPs that possess the transglycosylase and DD-transpeptidase activities, explicitly exploring the cross-talk between the two active sites that would influence one another in biosynthesis of cell wall.